PGN-challenged offspring immunity.

<p>PGN = peptidoglycan. Statistical evaluation (two-way-ANOVA) of priming effects on the increase of phenoloxidase (PO) and antibacterial (AMP) activity (lysozyme activity equivalent, <i>Micrococcus luteus</i>) of <i>Manduca sexta</i> offspring from differently treated parents (naive, PBS, PGN) one day after offspring PGN treatment in 4^th instar larvae and 22-day-old pupae (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone-0063392-g002" target="_blank">Fig. 2</a>). Means ± SE of PO activities and antibacterial activities of PGN-challenged offspring individuals are shown in Table S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>. Statistical evaluation of priming effects on the increase of immunity after offspring immune challenge by PBS is shown in Table S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>.</p><p>Data were Box-Cox transformed prior to analysis in order to reach normal distribution PO = PÔ0.318, AMP = AMPˆ0.046. Significant <i>P</i>-levels are shown in bold.</p

Unchallenged offspring immunity.

<p>Statistical evaluation (two-way ANOVA) of the priming effects on phenoloxidase (PO) and antibacterial (AMP) activity (lysozyme activity equivalent, <i>Micrococcus luteus</i>) of unchallenged <i>Manduca sexta</i> offspring from differently treated parents (naive, PBS, PGN) (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone-0063392-g001" target="_blank">Fig. 1</a> and Tables S3, S4 for post hoc test data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>).</p><p>Data were Box-Cox transformed prior to analysis in order to reach normal distribution PO = PO^∧0.185, AMP = AMP^∧0.095. Significant <i>P</i>-levels are shown in bold.</p

Performance of unchallenged offspring.

<p>Statistical evaluation (Generalized linear model; post hoc <i>U</i>-test) of priming effects on the change of weight and developmental time of (unchallenged) <i>Manduca sexta</i> offspring due to parental treatment (naive, PBS, PGN) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone-0063392-g004" target="_blank">Fig. 4A</a>). Means ± SE are shown in Table S9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>. Significant <i>P</i>-levels are shown in bold.</p

Reproductive fitness of <i>Manduca sexta</i> females in the parental and offspring generation.

<p>Total number of eggs laid by females of the differently treated parental generations and by the resulting (untreated) offspring generation. Female and male parents received a priming treatment in their pupal stage: Naive) untreated, PBS) control-injected with phosphate buffered saline, PGN) injected with peptidoglycan. Untreated offspring females mated with untreated males that were originating from parents subjected to the same parental priming treatment. Means ± SE are given. <i>N = </i>8 individuals from each parental group. Statistics: 2-way-ANOVA, post hoc analysis Tukey-test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone-0063392-t005" target="_blank">Table 5</a>), Tukey-test: different letters indicate statistical differences (<i>P</i><0.01).</p

Persistence of transgenerational immune priming effects on the increase of immune activity of <i>Manduca sexta</i> offspring after offspring immune challenge by PGN in 21-day-old pupae.

<p>A) Increase of phenoloxidase (PO) activity and B) increase of antibacterial (AMP) activity (lysozyme activity equivalent, <i>Micrococcus luteus</i>) were measured in 1-day-old and 3-day-old adults, i.e. three and five days, respectively, after offspring immune treatment in 21-day-old pupae. Female and male parents received a priming treatment in their pupal stage: Naive) untreated, PBS) control-injected with phosphate buffered saline, PGN) injected with peptidoglycan. If the symbol for offspring of naive parents is not visible, it is overlaid by another symbol. Increase of immune activity was measured as increase = (Activity after PGN treatment of the offspring)/(Mean activity of unchallenged offspring); value 1 is labelled by a line that indicates no change in immune activity after offspring challenge. Please note the comparable scales for increases which show the immunity and visualise the strong priming effects on offspring AMP activity, but the lack of effects on PO activity in the offspring. Mean values ± SE are given. <i>N = </i>9 individuals of each developmental stage from each parental group. Means of absolute data of PGN- and PBS-treated offspring are shown in Table S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>. Differences between the parental priming treatments and the time intervals after offspring PGN treatment were compared by two-way ANOVA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone-0063392-t003" target="_blank">Table 3</a>) and post hoc analysis Tukey tests (Table S7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>). Statistical evaluation of priming effects on the persistence of immunity after offspring immune challenge by PBS is shown in Table S8 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>.</p

Transgenerational immune priming effects on weight and developmental time of unchallenged <i>Manduca sexta</i> offspring.

<p>Change (increase/decrease) in weight (<b>A</b>) and developmental time (<b>B</b>) of offspring derived from naive parents and offspring of PBS- or PGN-treated parents were calculated for each offspring stage of each parental group as ratio = (Individual weight or developmental time of offspring of the respective parental treatment group)/(Mean weight or developmental time of offspring derived from naive parents). Larval weight was determined at the last day of each instar, whereas pupal and adult weight was determined at the first day of the respective stage. Value 1 is labelled by a line that indicates no change in weight or developmental time compared to offspring derived from naive parents. Female and male parents received a priming treatment in their pupal stage: Naive) untreated, PBS) control-injected with phosphate buffered saline, PGN) injected with peptidoglycan. If the symbol for offspring of naive parents is not visible, it is overlaid by another symbol. Mean ratios ± SE are given. <i>N = </i>18 individuals of each developmental stage except for adults <i>N</i> = 9 from each parental group. Means of absolute data of offspring weight and developmental time are shown in Table S9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>. Differences between the parental priming treatments and the offspring developmental stages were compared by Generalized linear model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone-0063392-t004" target="_blank">Table 4</a>) and post hoc analyses <i>U</i>-test (Table S10 weight, table S11 developmental time in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>).</p

Transgenerational immune priming effects on the increase of immune activity of <i>Manduca sexta</i> offspring larvae and pupae one day after offspring immune challenge by PGN.

<p>A) Increase of phenoloxidase (PO) activity and B) increase of antibacterial (AMP) activity (lysozyme activity equivalent, <i>Micrococcus luteus</i>) were measured in 4^th instar larvae and 22-day-old pupae one day after offspring immune treatment. Female and male parents received a priming treatment in their pupal stage: Naive) untreated, PBS) control-injected with phosphate buffered saline, PGN) injected with peptidoglycan. If the symbol for offspring of naive parents is not visible, it is overlaid by another symbol. Increase of immune activity was measured as increase = (Activity after PGN treatment of the offspring)/(Mean activity of unchallenged offspring); value 1 is labelled by a line that indicates no change in immune activity after offspring challenge. Please note the comparable scales for increases which show the immunity and visualise the strong priming effects on offspring AMP activity, but the lack of effects on PO activity in the offspring. Mean values ± SE are given. <i>N = </i>9 individuals of each developmental stage from each parental group. Means of absolute data of PGN- and PBS-treated offspring are shown in Table S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>. Differences between the parental priming treatments and the offspring developmental stages were compared by 2-way-ANOVA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone-0063392-t002" target="_blank">Table 2</a>). Statistical evaluation of priming effects on the increase of immunity after offspring immune challenge by PBS is shown in Table S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063392#pone.0063392.s001" target="_blank">File S1</a>.</p

Cuticular hydrocarbon phenotypes of indivudual Phaedon beetles

Relative composition of cuticular hydrocarbons (% total peak area) of individual 21-d-old beetles of Phaedon cochleariae and P. armoraciae. Beetles were either reared on their native host plant species or on Brassica rapa ssp. pekinensis

Responses from male and female receptor neurons to vinegar and its main components. A.

<p>Representative traces of extracellular recordings from ab1, ab2, and ab3 sensilla in response to the odour of apple cider vinegar (10% dilution in distilled water). Action potentials fired by specific neurons are identified as A, B, C or D. Stimulus duration (500 ms) is indicated by the black bar. <b>B.</b> Deletion of either A or B neurons in ab1 sensilla confirms that both ab1A and ab1B neurons responded to apple cider vinegar with ab1A dominating. A Diphtheria toxin transgene (UAS-DTi) was expressed by using the Or42b-Gal4 (A) or Or92a-Gal4 (B) driver to ablate neurons. <b>C.</b> Responses from neurons in ab1, ab2, and ab3 sensilla of male and female flies to a range of vinegar dilutions (in distilled water) applied to filter paper. Values are means ± SEM. Most SEM are too small to be seen (N_males = 3–11, N_females = 4–9). <b>D.</b> Responses to the main components of apple cider vinegar diluted in distilled water at concentrations equivalent to 50% vinegar. Values are means ± SEM (N = 8–19).</p

Behavioural responses of flies to vinegar and CO₂. A.

<p>Schematic drawing of the two-trap cage assay used to assess <i>Drosophila</i> olfactory behaviour. A controlled airflow (white arrows) delivered through two bubble vials entered traps in a 30×30×30 cm cage. The traps consisted of a glass flask closed with a silicon plug and a plastic funnel by which the odour is released and flies can enter. About 100 flies (males and females 50∶50) were tested for 5 hours in darkness except for a small light below the opaque floor of the cage. A small controlled flow of CO₂ or pressurised air could be added to one of the traps (black arrow). <b>B.</b> No choice assays. Percentage of flies trapped in the two flasks when both either contained 3 ml apple cider vinegar, 3 ml distilled water, were empty or empty without airflow. Different letters above the bars denote significant differences (Kruskal-Wallis test, p<0.05; post-hoc Mann–Whitney U-test with Bonferroni correction, p<0.05). Values are medians over 10 replicates and error bars indicate the 25% and 75% percentiles. <b>C, D, E.</b> Two-choice assays. The numbers of flies trapped in the control trap are indicated on the left, those in the test trap on the right. Adding the two bars indicates the total number of flies trapped. <b>C.</b> Numbers of flies trapped in two-choice situations between water (W) and either apple cider vinegar (V), water with 1% pressurised air (A) or 1% CO₂ (C). <b>D.</b> Numbers of flies trapped in two-choice situations between apple cider vinegar alone (V) and vinegar mixed with either 1% pressurized air (A) or different concentrations of CO₂ (C). <b>E.</b> Percentage of flies trapped when males and females were tested separately (100 males or 100 females). Asterisks in C, D and E indicate a significant difference between the number of flies caught in the two traps (Friedman-ANOVA, p<0.05; followed by Wilcoxon signed rank test with sequential Bonferroni correction, p<0.05).</p